Cell capture system and method of use

ABSTRACT

A cell capture system including an array, an inlet manifold, and an outlet manifold. The array includes a plurality of parallel pores, each pore including a chamber and a pore channel, an inlet channel fluidly connected to the chambers of the pores; an outlet channel fluidly connected to the pore channels of the pores. The inlet manifold is fluidly connected to the inlet channel, and the outlet channel is fluidly connected to the outlet channel. A cell removal tool is also disclosed, wherein the cell removal tool is configured to remove a captured cell from a pore chamber.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/929,427, filed 15 Jul. 2020, which is a continuation of U.S. patentapplication Ser. No. 16/924,492, filed 9 Jul. 2020, which is acontinuation of U.S. patent application Ser. No. 16/835,603, filed 31Mar. 2020, which is a continuation of U.S. patent application Ser. No.16/679,639, filed 11 Nov. 2019, which is a continuation of U.S. patentapplication Ser. No. 16/599,704, filed 11 Oct. 2019, which is acontinuation of U.S. patent application Ser. No. 16/536,155, filed 8Aug. 2019, which is a continuation of U.S. patent application Ser. No.16/513,580, filed 16 Jul. 2019, which is a continuation of U.S. patentapplication Ser. No. 16/048,104, filed 27 Jul. 2018, which is acontinuation of U.S. patent application Ser. No. 15/657,553, filed 24Jul. 2017, which is a continuation of U.S. patent application Ser. No.15/333,420, filed 25 Oct. 2016, which is a is a continuation of U.S.patent application Ser. No. 14/607,918, filed 28 Jan. 2015, which is acontinuation of U.S. patent application Ser. No. 13/557,510, filed 25Jul. 2012, and claims the benefit of U.S. Provisional Application Ser.No. 61/513,785 filed on 1 Aug. 2011, which are all incorporated in theirentirety by this reference.

TECHNICAL FIELD

This invention relates generally to the particle analysis field, andmore specifically to a new and useful cell sorting and analysis systemwithin the cell sorting field.

BACKGROUND

With an increased interest in cell-specific drug testing, diagnosis, andother assays, systems that allow for individual cell isolation,identification, and retrieval are becoming more desirable within thefield of cellular analysis. Furthermore, with the onset of personalizedmedicine, low-cost, high fidelity cellular sorting systems are becominghighly desirable. However, preexisting cell capture systems suffer fromvarious shortcomings that prevent widespread adoption for cell-specifictesting. For example, flow cytometry requires that the cell besimultaneously identified and sorted, and limits cell observation to asingle instance. Flow cytometry fails to allow for multiple analyses ofthe same cell, and does not permit arbitrary cell subpopulation sorting.Conventional microfluidic devices rely on cell-specific antibodies forcell selection, wherein the antibodies that are bound to themicrofluidic device substrate selectively bind to cells expressing thedesired antigen. Conventional microfluidic devices fail to allow forsubsequent cell removal without cell damage, and only capture the cellsexpressing the specific antigen; non-expressing cells, which could alsobe desired, are not captured by these systems. Cellular filters canseparate sample components based on size without significant celldamage, but suffer from clogging and do not allow for specific cellidentification, isolation, and retrieval.

Thus, there is a need in the cell sorting field to create a new anduseful cell capture and analysis system.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the cell capture system.

FIG. 2 is a perspective view of a variation of the cell capture system.

FIGS. 3A, 3B, 3C, 3D, and 3E are schematic representations of a first,second, third, fourth, and fifth pore variation, respectively.

FIG. 4 is a top view of a variation of the cell capture system.

FIG. 5 is a top view of a second variation of the cell capture system.

FIG. 6 is a top view of a third variation of the cell capture system.

FIG. 7 is a top view of a fourth variation of the cell capture system.

FIG. 8 is a top view of a fifth variation of the cell capture system.

FIG. 9 is a top view of a variation of the cell capture system includingan isolation mechanism.

FIGS. 10A, 10B, and 10C are a schematic representation of introducing anisolation material, creating a unique photomask, and selecting for cellsof interest, respectively.

FIGS. 11A, 11B, 11C, and 1D are side views of a first, second, third andfourth optical element, respectively.

FIGS. 12A, 12B, 12C, 12D, 12E, and 12F is a schematic representation ofa method of cell capture system manufacture.

FIG. 13 is a schematic representation of a second method of cell capturesystem manufacture.

FIGS. 14A and 14B are a perspective view and a side view of a firstvariation of the cell removal tool, respectively.

FIGS. 15A and 15B are a schematic representation of a method ofmanufacture for a first variation of the cell removal tool.

FIGS. 16A, 16B, 16C, and 16D are schematic representations of a firstvariation of cell removal, including cell of interest identification,cell removal tool alignment, cell removal tool perforation of the toplayer, and cell of interest removal, respectively.

FIGS. 17A and 17B are schematic representations of a second variation ofcell removal, including cell of interest identification and cell removaltool alignment with the pore containing the cell of interest,respectively.

FIG. 18 is a top view of a pore including a variation of microspheres.

FIG. 19 is a variation of cell capture system use, including samplepreparation.

FIG. 20 is a schematic representation of an integrated platform withwhich the cell capture system can be used.

FIG. 21 is a schematic representation of a fluidic manifold.

FIG. 22 is a schematic representation of a sample workstation.

FIGS. 23A, 23B, and 23C are schematic representations of a method ofautomated focusing.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of the preferred embodiments of the inventionis not intended to limit the invention to these preferred embodiments,but rather to enable any person skilled in the art to make and use thisinvention.

As shown in FIGS. 1 and 2, the cell capture system 100 includes an array200, an inlet manifold 300, and an outlet manifold 400. The array 200includes a plurality of pores 220, each pore 220 including a chamber 222fluidly connected to a pore channel 224; an inlet channel 240 fluidlyconnected to the chamber 222; and an outlet channel 260 fluidlyconnected to the pore channel 224. The inlet manifold 300 is preferablyfluidly coupled to the inlet channel 240, and the outlet manifold 400 ispreferably fluidly coupled to the outlet channel 260. The cell capturesystem 100 functions to isolate, capture, and hold cells, morepreferably single cells, at known, addressable locations. Once cells arecaptured in defined locations determined by single cell capturechambers, the fluidic network can be used to provide and delivermultiple reagents simultaneously or sequentially to enable a variety ofcellular, sub-cellular or molecular reactions to be performed in each ofthe single cells. The cell capture system 100 can also allow opticalinterrogation and detection of events on each of the captured cells at asingle cell level. The cell capture system 100 can additionally functionto selectively release or facilitate selective removal of one or more ofthe captured cells. The cell capture system 100 can confer the benefitsof real-time cell tracking, viable cell retrieval, and selectivedownstream molecular testing, either in the same microfluidic chip oroff-chip. The cell capture system 100 can be used to capture circulatingtumor cells (CTCs), but can alternatively be used to capture any othersuitable cell of possible interest. The cell capture system 100 ispreferably defined on a chip, more preferably a microfluidic chip, butcan alternatively be located on or defined by any suitable substrate110.

The cell capture system 100 preferably achieves individual cell captureand retention without antibody coated chambers 222, and preferablymaintains the viability of the cells throughout isolation, capture,retention, and removal. The cell capture system 100 preferablyadditionally minimizes clogging. The cell capture system 100 preferablyaccomplishes this by utilizing suitably sized pores 220 and byleveraging massively parallel flow, such that the cells near the sampleinlet 320 preferably experience substantially the same pressure as thecells distal the sample inlet 320 while minimizing the total pressuredifferential required to flow liquid at high rates through the cellcapture system. The variation in pressure felt by cells at therespective ends of the array is preferably less than 50% or 75% of theinlet pressure, but can alternatively be more or less. The sample flowis preferably substantially laminar, but can alternatively have anyother suitable flow characteristics. The sample flow path is preferablysubstantially unidirectional, but can alternatively be bi-directional.Cell sorting and viability maintenance can additionally be accomplishedby controlling the sample flow rate through the system, or through anyother suitable means.

In operation, the cell capture system 100 preferably receives a sampleunder positive pressure through the inlet manifold 300. Sample flowthrough the cell capture system 100 can be additionally or alternativelyencouraged by providing negative pressure at the outlet manifold 400.Alternatively, actuation pressure may be cycled in a pulse-widthmodulation fashion or sinusoidal fashion to provide net actuationpressure, either net positive at the inlet or net negative at theoutlet. The sample preferably flows through the inlet manifold 300 tothe inlet channel 240, through the chambers 222 and pore channels 224 tothe outlet channel 260, and out of the cell capture system 100 throughthe outlet manifold 400. Cells of a predetermined size are preferablytrapped within the chamber 222 as the sample flows through the pores220, wherein the pore channel 224 dimensions preferably prevent flow ofcertain cell sizes therethrough. For example, in the variation of thecell capture system 100 configured to capture CTCs, the chambers 222 arepreferably dimensioned larger than a CTC, and the pore channels 224 arepreferably dimensioned smaller than the CTC.

As shown in FIGS. 1 and 2, the array 200 of the cell capture system 100functions to capture cells of interest in addressable, known locations.The array 200 includes a plurality of pores 220, each pore 220 includinga chamber 222 fluidly connected to a pore channel 224; an inlet channel240 fluidly connected to the chamber 222; and an outlet channel 260fluidly connected to the pore channel 224. The array 200 is preferablysubstantially linear with a substantially constant width, but canalternatively be nonlinear and/or have a variable width. The array 200preferably includes a linear inlet channel 240, a linear outlet channel260 arranged parallel to the inlet channel 240, and a plurality ofparallel pores 220 arranged therebetween, normal to the inlet 320 andoutlet channels 260. However, the array 200 can alternatively besubstantially linear with a diverging or converging width, wherein thelinear inlet 320 and outlet channels 260 are arranged at an angle, andconsecutive pores 220 have increasing or decreasing lengths. The array200 can alternatively be serpentine, boustrophedonic, or have any othersuitable geometry.

The cell capture system 100 preferably includes one or more arrays 200.More preferably, the cell capture system 100 includes multiple arrays200 aligned in parallel, such that the outlet channel 260 of a firstarray 200 is preferably oriented parallel to the inlet channel 240 of anadjacent array 200. The multiple arrays 200 are preferably substantiallyidentical, wherein the pores 220 of the multiple arrays 200 preferablyhave the same or similar chamber 222 dimensions and pore channel 224dimensions, the inlet channels 240 preferably have similar lengths andwidths, and the outlet channels 260 preferably have similar lengths andwidths. However, different arrays 200 within the cell capture system 100can have different pore 220 characteristics, different inlet channel 240characteristics, and/or different outlet channel 260 characteristics.For example, a cell capture system 100 can include multiple arrays 200,wherein a first array 200 has pores 220 with a large pore channel 224width that captures large cells, a second array 200 has pores 220 with amedium pore channel 224 width that captures medium sized cells, and athird array 200 has pores 220 with a small pore channel 224 width thatcaptures small cells.

The multiple arrays 200 are preferably fluidly coupled in parallel bythe inlet manifold 300. Alternatively, the multiple arrays 200 can befluidly coupled in series, as shown in FIG. 8, wherein the outletchannel 260 of an upstream array 200 feeds into the inlet channel 240 ofan adjacent downstream array 200.

The pores 220 of the array 200 function to capture and retain cells.More preferably, the pores 220 of the array 200 capture and retain asingle cell. The pores 220 preferably include a chamber 222 configuredto hold a cell, and a pore channel 224 fluidly connected to the chamber222. The chamber 222 preferably has a length that prevents cell egressdue to crossflow within the inlet channel 240, and a width or a depththat prevents excessive cell movement but allows for the cell to moveenough such that the cell does not block the pore channel junction. Theend of the pore channel 224 proximal the chamber 222 preferably has awidth that prevents the cell of interest 10 from passing through, whilepermitting smaller sample component (e.g. lysed cells, cellularcomponents, etc.) flow therethrough. The end of the pore channel 224proximal the chamber 222 is preferably smaller than the diameter of thecell of interest 10, but can have any other suitable dimension.

Each array 200 preferably includes multiple pores 220. For example, anarray 200 can include 100, 1000, 10,000, 1,000,000, or any suitablenumber of pores 220. The pores 220 are preferably fluidly coupled inparallel within the array 200, but can alternatively be fluidly coupledin series within the array 200. The pores 220 are preferably arranged inparallel within the array 200, wherein the longitudinal axes of adjacentpores 220 are preferably parallel. However, the pores 220 can bearranged at an angle to adjacent pores 220 within the array 200. Thepores 220 of a given array 200 are preferably substantially similar oridentical, with chambers 222 of substantially the same dimension andpore channels 224 of substantially the same dimension. However, a singlearray 200 can have pores 220 with substantially different chamber 222and pore channel 224 dimensions, with varying chamber 222 lengths,chamber 222 widths, chamber 222 depths, pore channel 224 lengths, porechannel 224 widths, pore channel 224 depths, number of pore channels 224per pore 220, number of chambers 222 per pore 220, or pores 220 thatvary along any other suitable parameter. For example, an array 200 canhave multiple pores 220 arranged in parallel, wherein consecutive pores220 have decreasing pore channel widths.

The chamber 222 of the pore 220 functions to retain a cell. The chamber222 is preferably fluidly connected to the inlet channel 240 and thepore channel 224. The chamber 222 preferably has a length and widthconfigured to retain an isolated cell, wherein the chamber 222 isdimensioned to prevent cell egress from the chamber 222 due to inletchannel cross-flow. In one variation, this is achieved by controllingthe width to height ratio of chamber 222. The width to height ratio ofthe chamber 222 is preferably 1, but can alternatively be 1.25, 0.5, orany other suitable ratio. The chamber 222 is preferably configured toretain a single cell and to prevent multiple cell retention. In onevariation, the chamber 222 is dimensioned such that the height/width ofthe chamber 222 prevents a second cell from settling to the end of thechamber 222 proximal the pore channel 224 (e.g. the bottom of thechamber 222), and the length of the chamber 222 prevents a single cellegress from the chamber 222 (e.g. the length is longer than the celldiameter), but encourages egress of a second cell from the chamber 222(e.g. the length is longer than the cell diameter, but shorter than twocell diameters). However, the chamber 222 can be configured to retainmultiple cells. The chamber 222 preferably has a length, width and depthbetween 5-200 microns, but can alternatively have any suitabledimensions. In one variation, the chamber has a length of 50micrometers, a width of 50 micrometers, and a height of 50 micrometers.In another variation, the chamber has a length of 25 micrometers, awidth of 25 micrometers, and a height of 30 micrometers. The chamber 222preferably has a substantially constant cross-section, but canalternatively have a tapering cross-section, preferably tapering fromthe inlet channel 240 to the pore channel 224. The variablecross-section can be the cross-section parallel to the broad face of thesubstrate 112 and/or the cross-section perpendicular to the longitudinalaxis of the chamber 222. In one variation, as shown in FIG. 3B, thechamber 222 has a rectangular cross-section, wherein the pore channel224 connects to a side of the chamber 222 opposing that connected to theinlet channel 240. In another variation, the chamber 222 has a paraboliccross section, as shown in FIG. 3A and FIG. 3C, wherein the pore channel224 connects to the apex of the parabolic profile. In another variation,as shown in FIG. 3D, the chamber cross section linearly decreases fromthe inlet channel 240 to the pore channel 224. In another variation, asshown in FIG. 3E, the chamber cross-section decreases stepwise from theinlet channel 240 to the pore channel 224. In this variation, thechamber 222 defines multiple sub-chambers, wherein the multiplesub-chambers are preferably fluidly connected in series, wherein a firstsub-chamber is fluidly connected to the inlet channel 240 and the lastsub-chamber is fluidly connected to the pore channel 224. The firstsub-chamber preferably has the largest width and/or depth, and the lastsub-chamber preferably has the smallest width and/or depth. Thetransition between the inlet channel 240 and the chamber 222 preferablyexhibits a convex angle (e.g. a 900 angle), but can alternatively becurved as shown in FIG. 3C. The transition between the chamber 222 andthe pore channel 224 preferably also exhibits a convex angle (e.g. a 90°angle), but can alternatively be curved.

The pore channel 224 of the pore 220 functions to filter out the cell ofinterest 10 and to allow smaller sample components to flow through. Thepore channel 224 is preferably fluidly connected to the chamber 222 andthe outlet channel 260. More preferably, the pore channel 224 is fluidlyconnected to the portion of the chamber 222 distal from the inletchannel 240. The pore channel 224 is preferably substantially straightand linear, but can alternatively be curved. The pore channel 224preferably has a width smaller than the diameter of the cell of interest10, such that the pore channel 224 prevents cell passage therethrough.The pore channel 224 preferably has a width and depth between 1-25microns and a length between 5-500 microns, but can have any othersuitable width, depth, and length. In one variation, the pore channel224 has a width of 7-10 micrometers, a depth of 7-10 micrometers, and alength of 5-50 micrometers. The pore channel 224 preferably has asubstantially constant cross-section, but can alternatively have atapering or variable cross section. The pore channel 224 is preferablyaligned with its longitudinal axis parallel the longitudinal axis of thechamber 222. More preferably, the pore channel 224 is coaxial with thechamber 222. However, the pore channel 224 can be aligned at an anglewith the chamber 222. Each pore 220 preferably includes a single porechannel 224, but can alternatively include multiple pore channels 224,wherein the multiple pore channels 224 preferably extend in parallelfrom the end of the respective chamber 222 proximal the outlet channel260.

The inlet channel 240 of the array 200 functions to receive a volume ofthe sample and to distribute the sample to the pores 220. The inletchannel 240 preferably fluidly connects the inlet manifold 300 to thechambers 222 of the array 200. The inlet channel 240 preferably includesa first end, a second end, and a channel connecting the first and secondends. The inlet channel 240 is preferably fluidly connected to the inletmanifold 300 at the first end, is fluidly connected to the chambers 222of the array 200 along the inlet channel 240 length, and is preferablyfluidly sealed at the second end. The second end can be sealed by thesubstrate 110 or can be sealed by a sealant, such as a self-sealinglaminate (e.g. made of rubber, polyethylene, etc.). However, the inletchannel 240 can include a first and/or second valve disposed within thefirst and/or second end, wherein the valves can operate between an openand a closed state. The body of the inlet channel 240 is preferablydefined by the substrate 110, but can alternatively be partially definedby the substrate 110, wherein the other portions can be defined byself-sealing laminate or any other suitable sealant. The inlet channel240 is preferably arranged such that the inlet channel longitudinal axisis perpendicular to the longitudinal axes of the chambers 222, but canalternatively be arranged at an angle. The chambers 222 preferablyextend from a single side of the inlet channel 240, but canalternatively extend from multiple sides (e.g. opposing sides). Theinlet channel 240 is preferably substantially straight, but canalternatively be curved or bent. The inlet channel 240 preferably has asubstantially constant cross-section, but can alternatively have avariable cross section. The cross-section can be the cross-sectionparallel to the inlet channel longitudinal axis or perpendicular to theinlet channel longitudinal axis. In one variation, the inlet channel 240tapers with distance away from the inlet manifold 300. The inlet channel240 preferably has a depth and width larger than the diameter of thecell of interest 10. The inlet channel 240 preferably a depth and/orwidth between 5-200 microns, but can alternatively have any suitabledepth and/or width. In one variation, the inlet channel has a width of50-100 micrometers, and a depth of 50-100 micrometers. The inlet channel240 preferably has a length that can accommodate all the pores 220 ofthe array 200. In one variation, the inlet channel 240 preferably has alength longer than the combined widths of the chambers 222. In anothervariation, the inlet channel 240 extends to the edge of the substrate110. Each array 200 preferably includes one inlet channel 240, but canalternatively include multiple inlet channels 240. For example, an array200 can include two inlet channels 240 that feed two sets of pores 220extending from either side of a central outlet channel 260, wherein eachinlet channel 240 feeds one set of pores 220. However, the array 200 caninclude any suitable configuration of inlet channels 240.

The outlet channel 260 of the array 200 functions to receive a volume ofthe sample and to distribute the sample to the pores 220. The outletchannel 260 preferably includes a first end, a second end, and a channelconnecting the first and second ends. The outlet channel 260 ispreferably fluidly connected to the outlet manifold 400 at the secondend, fluidly connected to the chambers 222 of the array 200 along theoutlet channel 260 length, and is preferably fluidly sealed at the firstend. The first end of the outlet channel 260 can be sealed by thesubstrate 110 or can be sealed by a sealant, such as a self-sealinglaminate (e.g. made of rubber, polyethylene, etc.). Alternatively, theoutlet channel 260 can include a first and/or second valve disposedwithin the first and/or second end, wherein the valves can operatebetween an open and a closed state. The body of the outlet channel 260is preferably defined by the substrate 110, but can alternatively bepartially defined by the substrate 110, wherein the other portions canbe defined by self-sealing laminate or any other suitable sealant. Theoutlet channel 260 is preferably arranged such that the outlet channellongitudinal axis is perpendicular to the longitudinal axes of thechambers 222, but can alternatively be arranged at an angle. Thechambers 222 preferably extend from a single side of the outlet channel260, but can alternatively extend from multiple sides (e.g. opposingsides). The outlet channel 260 is preferably substantially straight, butcan alternatively be curved or bent. The outlet channel 260 preferablyhas a substantially constant cross-section, but can alternatively have avariable cross section. The outlet channel 260 cross-section can be thecross-section parallel outlet channel longitudinal axis or perpendicularthe outlet channel longitudinal axis. In one variation, the outletchannel 260 tapers with distance away from the outlet manifold 400. Theoutlet channel 260 preferably has a depth and width similar to that ofthe inlet channel 240, but can alternatively have a depth and widthsmaller or larger than that of the inlet channel 240. The outlet channel260 preferably a depth and/or width between 5-200 microns, but canalternatively have any suitable depth and/or width. In one variation,the outlet channel has a width of 50-100 micrometers, and a depth of50-100 micrometers. The outlet channel 260 preferably has a length thatcan accommodate all the pores 220 of the array 200. In one variation,the outlet channel 260 preferably has a length longer than the combinedwidths of the chambers 222. In another variation, the outlet channel 260extends to the edge of the substrate 110. Each array 200 preferablyincludes one outlet channel 260, but can alternatively include multipleoutlet channels 260. For example, an array 200 can include two outletchannels 260 that egress two sets of pores 220 extending from eitherside of a central inlet channel 240, wherein each outlet channel 260egresses one set of pores 220.

The inlet manifold 300 of the cell capture system 100 functions toreceive a sample and to distribute the sample to the arrays 200. Morepreferably, the inlet manifold 300 distributes the sample to an inletchannel 240 of an array 200. The inlet manifold 300 preferablyadditionally includes an inlet 320, wherein the inlet manifold 300receives the sample from the inlet 320. The inlet manifold 300preferably provides a substantially linear flow path from the inlet 320to the inlet channels 240 while substantially minimizing the differencesin pressure experienced by different arrays 200 within the system. Theinlet manifold 300 is preferably defined within the same substrate broadface as the array 200, but can alternatively be defined through aportion or the entirety of the substrate thickness. The entirety of theinlet manifold 300, except for the inlet 320, is preferably fluidlysealed by the top layer 120.

In one variation, as shown in FIG. 4, the cell capture system 100includes multiple inlet manifolds 300, one for each inlet channel 240.In this variation, the multiple inlet manifolds 300 can receive a singlesample or multiple samples.

In another variation, as shown in FIGS. 5, 6, and 7, the system includesa single inlet manifold 300 that feeds all the inlet channels 240. Theinlet manifold 300 preferably fluidly connects the arrays 200 inparallel to facilitate parallel flow throughout the cell capture system100. However, the inlet manifold 300 can alternatively fluidly connectthe arrays 200 in series or in any suitable combination of series andparallel flow. The inlet manifold 300 preferably includes one or moretiers of inlet sub-manifolds 302. Each inlet sub-manifold 302 preferablyincludes a main channel 204 and a plurality of feeder channels 206,wherein the feeder channels 206 facilitate sample flow into subsequentsub-manifolds or the inlet channels 240 of the arrays 200. The feederchannels 206 directly fluidly connected to the inlet channels 240 arepreferably aligned and coextensive with the inlet channels 240, but canalternatively be perpendicular to the inlet channels 240 or arranged inany suitable configuration. The main channel 204 preferably fluidlyconnects the feeder channels 206 in parallel. The feeder channels 206are preferably arranged parallel to the other feeder channels 206, andpreferably all extend perpendicularly from one side of the main channel204. However, the feeder channels 206 can be arranged at an acute anglerelative to the main channel 204, extend from opposing sides of the mainchannel 204, or be otherwise suitably arranged. The sub-manifoldsdirectly fluidly connected to the inlet channels 240 are preferably eachcoupled to a subset of the arrays 200 to minimize the pressuredifference between the arrays 200 proximal the sub-manifold inlet andthe arrays 200 distal the sub-manifold inlet 320. However, a singlesub-manifold can directly feed all the arrays 200 of the cell capturesystem 100.

In one variation, the cell capture system 100 includes an inlet manifold300 with one inlet sub-manifold tier, wherein the inlet sub-manifold 302includes multiple feeder channels 206, each feeder channel independentlyfluidly connected to a inlet channel 240 of an array 200.

In another variation, the cell capture system 100 includes an inletmanifold 300 including two tiers of inlet sub-manifolds 302 (as shown inFIG. 5), wherein the feeder channels 206 of the first tier are fluidlyconnected to the main channels 204 of the second tier, and the feederchannels 206 of the second tier are fluidly connected to the inletchannels 240. The first tier preferably includes one inlet sub-manifold302, with one main channel 204 and multiple feeder channels 206. Thesecond tier preferably includes multiple inlet sub-manifolds 302,wherein each second tier inlet sub-manifold 302 is fluidly connected toa first tier feeder channel and a subset of the arrays 200 of the cellcapture system 100. For example, a second tier inlet sub-manifold 302can be fluidly connected to four inlet channels 240 of a forty-array 200cell capture system 100, wherein the second tier inlet sub-manifold 302includes one main channel 204 and four feeder channels 206, each feederchannel independently fluidly connected to an inlet channel 240. In thisvariation, the first tier main channel 204 preferably has a larger widthand/or height than the second tier main channels 204, and the first tierfeeder channels 206 preferably have a larger width and/or height thanthe second tier feeder channels 206. The second tier feeder channels 206are preferably substantially the same width and/or height as the inletchannels 240, but can alternatively have different dimensions than theinlet channels 240. In another variation, the inlet manifold 300includes three tiers of branching inlet sub-manifolds 302. However, theinlet manifold 300 can include any suitable number of inlet sub-manifoldtiers.

The inlet 320 of the inlet manifold 300 functions to provide a fluidconnection between the cell capture system 100 exterior and interior.More preferably, the inlet 320 provides a fluid connection between thecell capture system 100 exterior and the inlet manifold 300. The cellcapture system 100 preferably includes one inlet 320, but canalternatively include multiple inlets 320. Each inlet 320 is preferablyfluidly connected to one inlet manifold 300 through a fluid connection(e.g. a channel), but can alternatively be connected to multiple inletmanifolds 300. Each inlet manifold 300 is preferably fluidly connectedto one inlet 320, but can alternatively be connected to multiple inlets320. The longitudinal axis of the inlet 320 is preferably normal to thelongitudinal axis of the main channel 204 of the inlet manifold 300, butcan alternatively be parallel. The longitudinal axis of the inlet 320 ispreferably normal to the broad face of the substrate 112, but canalternatively be parallel to the broad face of the substrate 112, at anangle to the broad face of the substrate 112, or arranged in anysuitable manner. In one variation of the cell capture system 100, theinlet 320 is a hole or aperture through a portion of the substratethickness, extending from a broad face of the substrate 112 to the planedefining the inlet manifold 300. The broad face of the substrate 112from which the inlet 320 extends can either be the broad face on whichthe inlet manifold 300 is defined, wherein a fluid connection connectingthe inlet 320 and the inlet manifold 300 is also defined on the samebroad face, or can be the broad face opposite that on which the inletmanifold is defined 114, wherein the inlet 320 extends throughsubstantially the whole of the substrate thickness to connect with theinlet manifold 300. In another variation of the cell capture system 100,the inlet 320 is a hole or aperture through a side of the substrate 110,wherein the inlet 320 extends in parallel with a broad face of thesubstrate 112 towards the inlet manifold 300. In this variation, a fluidconnection normal to the broad face of the substrate 112 preferablyconnects the inlet 320 with the inlet manifold 300. However, anysuitable configuration of the inlet 320 can be used.

The outlet manifold 400 of the cell capture system 100 functions toreceive filtered sample and to egress the filtered sample from the cellcapture system 100. More preferably, the outlet manifold 400 receivesthe filtered sample from an outlet channel 260 of an array 200. Theoutlet manifold 400 preferably additionally includes an outlet 420,wherein the outlet manifold 400 egresses the filtered sample from theoutlet 420. The outlet manifold 400 preferably provides a substantiallylinear flow path from the outlet channels 260 to the outlet 420, but canalternatively provide a tortuous flow path. The outlet manifold 400 ispreferably defined within the same substrate broad face as the array200, but can alternatively be defined through a portion or the entiretyof the substrate thickness, on the opposing broad face of the substrate112, or on any suitable portion of the substrate 110. The entirety ofthe outlet manifold 400, except for the outlet 420, is preferablyfluidly sealed by the top layer 120.

In one variation, as shown in FIG. 4, the cell capture system 100includes multiple outlet manifolds 400, one for each outlet channel 260.In this variation, the multiple outlet manifolds 400 can receive asingle filtered sample or multiple filtered samples.

In another variation, as shown in FIGS. 5, 6, and 7, the system includesa single outlet manifold 400 that receives the filtered sample from allthe outlet channels 260. The outlet manifold 400 preferably fluidlyconnects the arrays 200 in parallel, but can alternatively fluidlyconnect the arrays 200 in series or in any suitable combination ofseries and parallel flow. The outlet manifold 400 preferably includesone or more tiers of outlet sub-manifolds 402. Each outlet sub-manifold402 preferably includes a main channel 204 and a plurality of feederchannels 206, wherein the feeder channels 206 facilitate filtered sampleflow from upstream sub-manifolds or the outlet channels 260 of thearrays 200 to the main channel 204. The feeder channels 206 directlyfluidly connected to the outlet channels 260 are preferably parallel andcoextensive with the outlet channels 260, but can alternatively beperpendicular to the outlet channels 260 or arranged in any suitableconfiguration. The main channel 204 preferably fluidly connects thefeeder channels 206 in parallel. The feeder channels 206 are preferablyarranged parallel to the other feeder channels 206, and preferably allextend perpendicularly from one side of the main channel 204. However,the feeder channels 206 can arranged at an acute angle relative to themain channel 204, extend from opposing sides of the main channel 204, orbe otherwise suitably arranged. The outlet sub-manifolds 402 directlyfluidly connected to the outlet channels 260 are preferably each coupledto a subset of the arrays 200. However, a single outlet sub-manifold 402can directly receive the filtered sample from all the arrays 200 of thecell capture system 100.

In one variation, the cell capture system 100 includes an outletmanifold 400 with one outlet sub-manifold tier, wherein the outletsub-manifold 402 includes multiple feeder channels 206, each feederchannel independently fluidly connected to a outlet channel 260 of anarray 200.

In another variation, the cell capture system 100 includes an outletmanifold 400 including two tiers of outlet sub-manifolds 402, whereinthe feeder channels 206 of the first tier are fluidly connected to themain channels 204 of the second tier, and the feeder channels 206 of thesecond tier are fluidly connected to the outlet channels 260. The firsttier preferably includes one outlet sub-manifold 402, with one mainchannel 204 and multiple feeder channels 206. The second tier preferablyincludes multiple outlet sub-manifolds 402, wherein each second tieroutlet sub-manifold 402 is fluidly connected to a first tier feederchannel and a subset of the arrays 200 of the cell capture system 100.For example, a second tier outlet sub-manifold 402 can be fluidlyconnected to four outlet channels 260 of a forty-array 200 cell capturesystem 100, wherein the second tier outlet sub-manifold 402 includes onemain channel 204 and four feeder channels 206, each feeder channelindependently fluidly connected to an outlet channel 260. In thisvariation, the first tier main channel 204 preferably has a larger widthand/or height than the second tier main channels 204, and the first tierfeeder channels 206 preferably have a larger width and/or height thanthe second tier feeder channels 206. The second tier feeder channels 206are preferably substantially the same width and/or height as the outletchannels 260, but can alternatively have different dimensions than theoutlet channels 260. In another variation, the outlet manifold 400includes three tiers of branching outlet sub-manifolds 402. In anothervariation, the outlet manifold 400 includes the same number of tiers asthe inlet manifold 300. However, the outlet manifold 400 can include anysuitable number of outlet sub-manifold tiers.

The outlet 420 of the outlet manifold 400 functions to provide a fluidconnection between the cell capture system 100 interior and the cellcapture system 100 exterior. More preferably, the outlet 420 provides afluid connection between the cell capture system 100 exterior and theoutlet manifold 400. The cell capture system 100 preferably includes oneoutlet 420, but can alternatively include multiple outlets 420. Eachoutlet 420 is preferably fluidly connected to one outlet manifold 400through a fluid connection (e.g. a channel), but can alternatively beconnected to multiple outlet manifolds 400. Each outlet manifold 400 ispreferably fluidly connected to one outlet 420, but can alternatively beconnected to multiple outlets 420. The longitudinal axis of the outlet420 is preferably normal to the longitudinal axis of the main channel204 of the outlet manifold 400, but can alternatively be parallel. Thelongitudinal axis of the outlet 420 is preferably normal to the broadface of the substrate 112, but can alternatively be parallel to thebroad face of the substrate 112, at an angle to the broad face of thesubstrate 112, or arranged in any suitable manner. In one variation ofthe cell capture system 100, the outlet 420 is a hole or aperturethrough a portion of the substrate thickness, extending from a broadface of the substrate 112 to the plane defining the outlet manifold 400.The broad face of the substrate 112 from which the outlet 420 extendscan either be the broad face on which the outlet manifold 400 isdefined, wherein a fluid connection connecting the outlet 420 and theoutlet manifold 400 is also defined on the same broad face, or the broadface opposite that on which the outlet manifold is defined 114, whereinthe outlet 420 extends through substantially the whole of the substratethickness to connect with the outlet manifold 400. When the inlet 320 isdefined on a broad face of the substrate 112, the outlet 420 ispreferably defined on the same broad face as the inlet 320, but canalternatively be defined on the opposing broad face. In anothervariation of the cell capture system 100, the outlet 420 is a hole oraperture through a side of the substrate 110, wherein the outlet 420extends in parallel with a broad face of the substrate 112 towards theoutlet manifold 400. In this variation, a fluid connection normal to thebroad face of the substrate 112 preferably connects the outlet 420 withthe outlet manifold 400. When the inlet 320 is also defined on a side ofthe substrate 110, the outlet 420 is preferably defined on a side of thesubstrate opposing the side defining the inlet 320. However, the outlet420 can alternatively be defined on the same side or an adjacent side.However, any suitable configuration of the outlet 420 can be used.

The cell capture system 100 can additionally include an isolationmechanism 500 that functions to isolate cells within individual pores220. In one variation, the isolation mechanism 500 includes an isolationinlet 520 and an isolation outlet 540, fluidly connected to an array200, that functions to permit isolation material ingress and egress,respectively. Both the isolation inlet 520 and the isolation outlet 540are preferably fluidly connected to both the inlet channel 240 and theoutlet channel 260. In one variation, as shown in FIG. 9, the isolationinlet 520 can be arranged between the first end of the inlet channel 240and the outlet channel 260 on the inlet end of the array 200, and theisolation outlet 540 is arranged between the second end of the inletchannel 240 and outlet channel 260 on the outlet end of the array 200.The isolation inlets 520 or outlets 540 of the arrays 200 can be fluidlyconnected in parallel or in series by one or more isolation inlet oroutlet manifolds, respectively. In operation, the isolation material ispreferably flowed through the isolation inlet 520, into the inletchannel 240 and outlet channel 260, to the isolation outlet 540, forminga first isolation layer between the chamber 222 and the inlet channel240, and a second isolation layer between the pore channel 224 and theoutlet channel 260. The isolation layers are preferably 10 to 20micrometers thick, but can alternatively be thicker. During isolationmaterial introduction, buffer is preferably simultaneously flowedthrough the inlet channel 240 and outlet channel 260, preferably in thesame direction as isolation material flow, wherein the buffer flow ratepreferably controls the thickness of the isolation material layers.Buffer flow is preferably established in the portions of the inlet 320and outlet channel 260 distal from the pores 220. The buffer flow rateis preferably maintained at laminar flow, but can alternatively have anyother suitable flow rate. Alternatively, the isolation inlet 520 andoutlet 540 can be fluidly connected to a first and second isolationchannel located within the inlet channel 240 and outlet channel 260,respectively, wherein the first and second isolation channel guidesisolation material flow. However, any other suitable mechanism that canestablish a first and second isolation layer can be used.

The isolation material preferably isolates a pore 220 within an array200. The isolation material preferably has a flow state and a set state,wherein a photochemical reaction, thermochemical reaction,polymerization reaction or any other suitable reaction switches theisolation material from the flow state to the set state. In the flowstate, the isolation material is preferably substantially viscous, suchthat the isolation material does not flow into the pores 220 duringintroduction into the cell capture system 100. In the set state, theisolation material is preferably a solid or gel that prevents cellegress from the pore 220, and is preferably porous or selectivelypermeable to permit buffer and reagent penetration therethrough. Theisolation material is preferably a photopolymerizable hydrogel, such asPEG or polyacrylamide with photoinitiator, but can alternatively be anysuitable material with any other suitable polymerization agent. In onevariation, the isolation layer may be an immiscible liquid such as oil.In another variation, select portions of the isolation material can bereacted to seal specific pores 220. For example, as shown in FIG. 10B aunique photomask 504 can be created that allows collimated irradiationof isolation material segments blocking pores 220 containing the cellsof interest. Photomask 504 may be created by high resolution printing ofUV-blocking black ink on a transparency sheet or by use of standardphotolithography on photoresist coated glass masks. The selective UVexposure of select regions of the microfluidic chip can also beaccomplished by moving a UV laser or a collimated and concentrated UVspot to the select locations using an x-y stage. As shown in FIG. 10C,undesired cells 20 and unreacted isolation material can then be removedfrom the cell capture system 100 by ingressing fluid through the outletmanifold 400 (e.g. backflowing). Alternatively, the photomask 504 canallow irradiation of isolation material segments blocking pores 220containing undesired cells 20, wherein desired cells 10 are retrievedfrom the system. However, any suitable portion of the isolation materialcan be reacted.

The cell capture system 100 can additionally include optical elements130 that function to facilitate imaging. The optical elements 130function to adjust incoming light, preferably to facilitate betterimaging. The optical elements 130 can function to bend, reflect,collimate, focus, reject, or otherwise adjust the incoming light. Theoptical elements 130 are preferably fabricated within the same processas the cell capture system 100 manufacture, but can alternatively beincluded after cell capture system 100 manufacture. The optical elements130 are preferably defined within the substrate 110, but canalternatively be defined by the top layer 120 or by a separatecomponent. Optical elements 130 can include light reflectors disposedwithin the substrate thickness adjacent the arrays 200 (as shown in FIG.11A), defined on a broad face of the substrate 112 opposite thatdefining the cell capture system 100 (as shown in FIG. 11B), ormicrolenses defined on the top layer 120 (as shown in FIG. 11C), lightcollimators, light polarizers, interference filters, 90° illumination,elements that minimize excitation rays from going into path of collectedfluorescence emission light, diffraction filters, light diffusers, orany other suitable optical element. Alternatively, the optical elements130 can be defined by an imaging stage (as shown in FIG. 11D) or by anyexternal component.

The cell capture system 100 can additionally include pore affinitymechanisms that function to attract a cell of interest 10 towards a porechamber 222. Pore affinity mechanisms can include electric field traps,features within the inlet channel 240 that direct flow into a pore 220,negative pressure application to the outlet channel 260, or any othersuitable pore affinity mechanism.

The cell capture system 100 is preferably defined on a substrate 110.More preferably, the cell capture system 100 is defined on a singlebroad face of a substrate 112, wherein the array 200, including theinlet channel 240, pores 220, and outlet channel 260, is preferablydefined on a single broad face of the substrate 112. More preferably,the array 200, inlet manifold 300, and outlet manifold 400 are alldefined on the same broad face. Thus, sample flow through the cellcapture system 100 preferably runs substantially parallel to the broadface of the substrate 112. The array 200, inlet manifold 300, and outletmanifold 400 are all preferably defined by recesses in the broad face ofthe substrate 112, but can alternatively be channels defined by wallsthat are built on top of the substrate 110, or defined in any othersuitable manner. The substrate 110 preferably defines a portion of thecell capture system 100 (e.g. three walls of the system), wherein theremaining portions (e.g. one wall) are preferably defined by a top layer120. The top layer 120 preferably forms a substantially fluidimpermeable seal with the substrate 110 to fluidly seal the cell capturesystem 100. Alternatively, the cell capture system 100 can be definedthrough the thickness of the substrate 110, wherein the inlet channel240 is defined on a first broad face of the substrate 112, the outletchannel 260 is defined on an opposing broad face of the substrate 112,and the pores 220 are defined through the thickness of the substrate110.

The substrate 110 is preferably optically transparent, biocompatible,and substantially inert. Examples of material that can be used for thesubstrate 110 include glass, high refractive index polymer, or any othersuitable optically transparent material; silicon; any suitable polymersuch as polyethylene, polypropylene, polycarbonate, acrylic, orsilicone; quartz, glass, metals, ceramics, or any other suitablematerial. The top layer 120 is preferably an optically clear layer thatis laminated, adhered, heat-bonded, laser-bonded, anodic bonded, orotherwise joined to the substrate 110. The top layer 120 is preferably apolymeric laminate, but can alternatively be a glass cover slip or anyother suitable top layer 120.

The cell capture system 100 is preferably manufactured throughmicrofabrication processes, but can alternatively be manufacturedthrough injection molding, a combination of microfabrication (e.g. tocreate masters) and injection molding (e.g. for bulk manufacturing), acombination of microfabrication (e.g. to create masters) and hotembossing (e.g. for bulk manufacturing), laser etching, CNC, or anyother suitable manufacturing process. Microfabrication techniques thatcan be used include photolithography, DRIE, wet etching, and anodicbonding, but any suitable microfabrication technique can be used. Thearrays 200, inlet manifold 300 and outlet manifold 400 are preferablyformed within a single manufacturing process, but can alternativelyformed through multiple sequential or interrupted processes. The inlet320 and outlet 420 can additionally be formed within the same process asthat of the arrays 200, inlet manifold 300, and outlet manifold 400, butcan alternatively be formed before or after using different processes.

In one variation, as shown in FIG. 12, the cell capture system 100 ismanufactured using an injection molding process. The injection moldingmaster includes an array-definition portion 102, a bottom-definitionportion 104, and one or more core pins 106. The array-definition portionpreferably includes the negative for the arrays 200, and canadditionally include the negative for the inlet manifold 300 and outletmanifold 400. The array-definition portion is preferably formed usingmicrofabrication techniques, but can alternatively be formed throughlaser cutting, CNC, or any other suitable method. The bottom-definitionportion preferably includes channels through which the core pins canextend. The core pins preferably have tapered ends that insert into thebottom-definition portion channels, and function to define the inlet 320and outlet 420. The substrate material is preferably injected from anedge of the cell capture system 100 or parallel to the broad face of theto-be substrate 110. However, the substrate material can be injectedthrough the bottom-definition portion, normal to the broad face of theto-be substrate 110, or through any other suitable portion of themaster.

In another variation, as shown in FIG. 13, the cell capture system 100is manufactured using a microfabrication process, and utilizes a seriesof photolithography steps to create the components of the cell capturesystem 100 on the substrate 110. However, the cell capture system 100can be formed using any other suitable method.

Examples of the Cell Capture System

In a first example, as shown in FIG. 4, the cell capture system 100includes a plurality of substantially identical arrays 200 arranged inparallel; a plurality of inlet manifolds 300, each independently fluidlyconnected to an inlet channel 240; a plurality of inlets 320, eachindependently fluidly connected to an inlet manifold 300; a plurality ofoutlet manifolds 400, each independently fluidly connected to an outletchannel 260; and a plurality of outlets 420, each independently fluidlyconnected to an outlet manifold 400. Each array 200 preferably includesa plurality of substantially identical pores 220 connected to an inletchannel 240 at the chamber 222 and an outlet channel 260 at the porechannel 224. The arrays 200, inlet manifolds 300, and outlet manifolds400 are preferably recesses defined on one broad face of a substrate112, and are preferably cooperatively defined by a top layer 120 thatfluidly seals the arrays 200, inlet manifolds 300, and outlet manifolds400 from the cell capture system 100 exterior. The inlets 320 andoutlets 420 are preferably holes defined through the thickness of thesubstrate 110, and preferably originate from the substrate broad faceopposing the face defining the arrays 200, inlet manifolds 300, andoutlet manifolds 400. Alternatively, the inlets 320 and outlets 420 canbe holes extending through the substrate 110 from the substrate sides.

In a second example, as shown in FIG. 5, the cell capture system 100includes a plurality of substantially identical arrays 200 arranged inparallel; one inlet manifold 300 including two or more tiers; an inlet320 fluidly connected to the inlet manifold 300; a plurality of outletmanifolds 400, each independently fluidly connected to an outlet channel260; and a plurality of outlets 420, each independently fluidlyconnected to an outlet manifold 400. Each array 200 preferably includesa plurality of substantially identical pores 220 connected to an inletchannel 240 at the chamber 222 and an outlet channel 260 at the porechannel 224. The inlet sub-manifolds 302 directly connected to the inletchannels 240 preferably each independently connect to ten or less inletchannels 240. For example, when the cell capture system 100 includesforty arrays 200, the cell capture system 100 preferably includes tensecond tier inlet sub-manifolds 302, each connected to four inletchannels 240. The arrays 200, inlet manifolds 300, and outlet manifolds400 are preferably recesses defined on one broad face of a substrate112, and are preferably cooperatively defined by a top layer 120 thatfluidly seals the arrays 200, inlet manifolds 300, and outlet manifolds400 from the cell capture system 100 exterior. The inlet 320 and outlets420 are preferably holes defined through the thickness of the substrate110, and preferably originate from the substrate broad face opposing theface defining the arrays 200, inlet manifolds 300, and outlet manifolds400. Alternatively, the inlet 320 and outlets 420 can be holes extendingthrough the substrate 110 from the substrate sides. Alternatively, theinlet 320 can be a hole defined through the thickness of the substrate110, while the outlets 420 are holes extending parallel to the substratebroad face through the substrate 110.

In a third example, as shown in FIGS. 7 and 8, the cell capture system100 includes a plurality of substantially identical arrays 200 arrangedin parallel; one inlet manifold 300 including two or more tiers; aninlet 320 fluidly connected to the inlet manifold 300; one outletmanifold 400 including two or more tiers; and an outlet 420 fluidlyconnected to the outlet manifold 400. Each array 200 preferably includesa plurality of substantially identical pores 220 connected to an inletchannel 240 at the chamber 222 and an outlet channel 260 at the porechannel 224. The outlet manifold 400 preferably has the same number oftiers as the inlet manifold 300, and preferably mirrors the inletmanifold 300. For example, an outlet sub-manifold 402 directly connectedto the arrays 200 is preferably connected to the same arrays 200 that acorresponding inlet sub-manifold 302 is directly connected to. However,the outlet manifold 400 can include a different number tiers, group thearrays 200 differently, or have any other suitable configuration. Theinlet 320 and outlet sub-manifolds 402 directly connected to the inlet320 and outlet channels 260 preferably each independently connect to tenor less inlet 320 and outlet channels 260, respectively. For example,when the cell capture system 100 includes forty arrays 200, the cellcapture system 100 preferably includes ten second tier inlet 320 andoutlet sub-manifolds 402, each connected to four inlet 320 and outletchannels 260, respectively. The arrays 200, inlet manifold 300, andoutlet manifold 400 are preferably recesses defined on one broad face ofa substrate 112, and are preferably cooperatively defined by a top layer120 that fluidly seals the arrays 200, inlet manifold 300, and outletmanifold 400 from the cell capture system 100 exterior. The inlet 320and outlet 420 are preferably holes defined through the thickness of thesubstrate 110, and preferably originate from the substrate broad faceopposing the face defining the arrays 200, inlet manifold 300, andoutlet manifold 400. Alternatively, the inlet 320 and outlet 420 can beholes extending through the substrate 110 from the substrate sides.Alternatively, the inlet 320 can be a hole defined through the thicknessof the substrate 110, while the outlet 420 is a hole extending parallelto the substrate broad face 112, through the substrate 110.

In a fourth example, as shown in FIG. 8, the cell capture system 100includes a plurality of different arrays 200 arranged in parallel butfluidly connected in series; one inlet manifold 300 connected to theupstream inlet channel 240; an inlet 320 fluidly connected to the inletmanifold 300; one outlet manifold 400 connected to the downstream outletchannel 260; and an outlet 420 fluidly connected to the outlet manifold400. The pore channel width of the arrays 200 preferably decreases witheach subsequent array 200 away from the inlet 320. Furthermore, thechamber size of the arrays 200 can decrease with each subsequent array200 away from the inlet 320. The inlet and outlet channel size of thearrays 200 can also decrease with each subsequent array 200 away fromthe inlet 320. Each array 200 preferably includes a plurality ofsubstantially identical pores 220 connected to an inlet channel 240 atthe chamber 222 and an outlet channel 260 at the pore channel 224. Theoutlet channel 260 of an upstream array 200 is preferably fluidlyconnected to the inlet channel 240 of the adjacent downstream array 200.In one specific example, the cell capture system 100 includes a first,second, third, and fourth array 200 fluidly connected in series. Thefirst array 200 has a pore channel size of 30 micrometers, the secondarray 200 has a pore channel size of 25 micrometers, the third array 200has a pore channel size of 15 micrometers, and the fourth array 200 hasa pore channel size of 10 micrometers. The arrays 200, inlet manifold300, and outlet manifold 400 are preferably recesses defined on onebroad face of a substrate 112, and are preferably cooperatively definedby a top layer 120 that fluidly seals the arrays 200, inlet manifold300, and outlet manifold 400 from the cell capture system 100 exterior.The inlet 320 and outlet 420 can be holes defined through the substrate110 on the same side of the substrate 110, on the same broad face of thesubstrate 112, on opposing broad faces of the substrate 110, on adjacentfaces of the substrate 110, or arranged in any suitable configuration.

In a fifth example, as shown in FIG. 9, the cell capture system 100includes a first and a second array set 202, each array set 202including a plurality of substantially identical arrays 200 arranged inparallel, wherein each array 200 preferably includes a plurality ofsubstantially identical pores 220. The cell capture system 100preferably includes one inlet manifold 300 fluidly connected to theinlet channels 240 of both array set 202 s, but can alternativelyinclude two inlet manifolds 300, each independently connected to anarray set 202, or any other suitable number of inlet manifolds 300. Inone variation, the inlet manifold 300 can be disposed between the arrayset 202 s, such that the second array set 202 is an enantiomer of thefirst array set 202. However, the inlet manifold 300 can be disposed inany suitable position. The cell capture system 100 preferably includesone inlet 320, but can alternatively include more. In one variation, theinlet 320 is arranged equidistant between the array set 202 s. The cellcapture system 100 preferably includes two outlet manifolds 400, one foreach array set 202, but can alternatively include a plurality of outletmanifolds 400, one manifold for each outlet channel 260, or any othersuitable number of outlet manifolds 400. In one variation, the outletmanifold 400(s) can be disposed proximal the substrate 110 edges, suchthat the outlet manifold 400(s) for the first array set 202 is arrangedon the side of the first array set 202 distal the second array set 202,and the outlet manifold 400(s) are arranged on the side of the secondarray set 202 distal the first array set 202. The cell capture system100 preferably includes at least two outlets 420, but can alternativelyinclude more.

In a sixth example, as shown in FIG. 9, the cell capture system 100 issubstantially similar to the cell capture system 100 of the fifthexample, and can additionally include a third array set 202 including aplurality of substantially identical parallel pores 220 and a retrievalchannel 502 fluidly connecting the inlet manifold 300 of the first andsecond array set 202 with the inlet manifold 300 of the third array set202. In this example, the third array set 202 can function as asingle-cell reactor, wherein each array 200 within the third array set202 can additionally include an isolation inlet 520 and an isolationoutlet 540 for each array 200 within the set, the isolation inlet 520and outlet 420 disposed between the first and second ends of the inletchannel 240 and outlet channel 260, respectively. In operation, theretrieval channel 502 is preferably sealed proximal the inlet manifold300, and cells of interest are isolated within the first and secondarray set 202 s by running a sample through the inlet 320, through inletmanifold 300, and into the first and second array set 202 s. After cellisolation, the retrieval channel 502 can be unsealed, the inlet 320sealed, and the isolated cells backflowed through the inlet manifold300, through the retrieval channel 502 and into the third array 200 byrunning a buffer through the outlet manifold 400(s) of the first andsecond array set 202 s. As shown in FIG. 10, the cells can then beisolated from adjacent cells by simultaneously introducing an isolatingmaterial, such as hydrogel, into the isolation inlet 520 and a bufferinto the first ends of the inlet channel 240 and outlet channel 260. Theisolating material is then preferably reacted to switch the isolatingmaterial from a flow state to a set state. In one variation, onlyportions of the isolating material sealing the pores 220 containing thecells of interest are reacted. For example, the isolated cells can bestained, the pores 220 containing the cells of interest identified (e.g.wherein the cells of interest emit a desired wavelength), and aphotomask 504 created, wherein the photomask 504 permits only theportions of the isolating material sealing the pores of interest to bephotoreacted (e.g. through UV irradiation). Unreacted isolating materialand undesired cells 20 can be egressed by backflowing buffer through theoutlet manifold 400. However, any other suitable method of selectiveisolating material reaction can be used. Reagents (e.g. flourogenicantibodies, etc), analytes, or any other suitable substance can beintroduced into the third array 200 through the third array inlet 320prior to cell isolation. Alternatively, reagents can be introduced postcell isolation. In a first variation, the reagents can be introducedthrough the third array inlet manifold, wherein the reagent penetratesthrough the set isolating material to ingress into the pore 220. In asecond variation, reagents, analytes, or other substances can beintroduced into individual pores 220 by introducing the substancethrough the portion of the top layer 120 contiguous with the pore ofinterest.

Cell Removal

The cell capture system 100 is configured to facilitate selective cellremoval from known, addressable locations. While an individual cell froma single pore 220 is preferably selectively removed, the system canfacilitate simultaneous multiple cell removal from a single array 200 ora subset of arrays 200. The cell is preferably removed by applying aremoval force to the cell. The removal force is preferably applied bypumping fluid through the pore channel 224 into the chamber 222, but canalternatively be applied by aspirating the contents out of the chamber222. In one variation, the pump pressure provided by a pump mechanism atthe cell capture system 100 outlet 420 is less than 10,000 Pa. In onespecific variation, the provided pump pressure is 6,000 Pa. However, anyother suitable pump or aspiration pressure can be used.

In a first variation of the cell removal method, one or more cells canbe removed from the cell capture system 100 by ingressing a purgingfluid through an outlet manifold 400 and collecting flushed-out cells atthe inlet 320 (backflowing the cells). This can be particularlydesirable when collection of cells from multiple fluidly linked sites isdesired. Cell capture system 100 s including multiple outlet manifolds400 (e.g. systems with one outlet manifold 400 per array 200) can beparticularly suited to this cell removal method, as the cells within agiven array 200 can be removed without affecting adjacent captured cellswithin other arrays 200 by only ingressing fluid through the outletmanifold 400 directly connected to the selected array 200.Alternatively, cell capture system 100 s with multiple tiers ofsub-manifolds can be suited to this cell removal method, wherein cellsretained within a subset of arrays 200 that are fluidly connected bysub-manifold can be simultaneously removed. However, any suitable cellcapture system 100 configuration can be utilized with this cell removalmethod.

In a second variation of the cell removal method, cell removal can beachieved by utilizing a cell removal tool 600. The cell removal tool 600of the cell capture system 100 functions to selectively remove one ormore isolated cells from an addressable location within the cell capturesystem 100. The cell removal tool 600 is preferably configured to removea cell from a single chamber 222, but can alternatively be configured tosimultaneously remove multiple cells from multiple chambers 222.

In a first variation of the cell removal tool, the cell removal tool 600is configured to puncture the top layer 120 from a direction normal tothe broad face of the substrate 112. The cell removal tool 600preferably removes the cell in a substantially normal direction from thebroad face of the substrate 112, but can alternatively remove the cellin an angled direction relative to the broad face of the substrate 112.The cell removal tool 600 preferably includes a hollow needle thatpunctures the top layer 120 and defines a substantially fluidly isolatedvolume in fluid communication with one or more pores 220 (e.g. thedesired number of pores 220). As shown in FIGS. 14A and 14B, the hollowneedle preferably includes one or more sealing elements at the tip 620,such as a polymeric coating or adequate geometry, that facilitate fluidseal formation with the top layer 120. The hollow needle preferablyincludes a cannula ending in a hollow tip 620. The cannula preferablydefines a lumen, and is preferably fluidly connected to a cellcollection volume. In one variation, the tip 620 includes geometry thatfacilitates fluid seal formation with the top layer 120. The tip 620preferably includes a first and second opposing wall, each havingconcave profiles that taper into a perforating end distal the cannula.The first wall is preferably an enantiomer of the second wall, but canalternatively be substantially identical or different. The first andsecond sides of each wall (622 and 624, respectively) preferably exhibitdifferent curvatures, such that the center of the perforating end ispreferably offset from the longitudinal axis of the lumen. However, thefirst and second walls can alternatively be substantially similar (e.g.have the same curvature). The first and second opposing walls preferablyfunction to perforate the top layer 120 and to form a first and secondfluid seal with the substrate 110 to define the fluidly isolated volume.However, the hollow needle can include any other suitable geometry. Inone variation, the hollow needles have a height of 200 micrometers and alumen diameter of 40 micrometers.

The hollow needle is preferably configured to form a substantiallyfluidly isolated volume within a pore chamber 222 of interest or asegment of the inlet channel 240 adjacent a pore chamber 222 ofinterest. A low-pressure generator (e.g. a pump) is preferably then usedto aspirate the retained cell out of the pore chamber 222, through thehollow needle, and into the cell collection volume.

The hollow needle is preferably manufactured using microfabricationtechniques, but can alternatively be injection molded, laser cut,stamped, or manufactured using any other suitable manufacturingtechnique. In one variation of hollow needle manufacture, as shown inFIG. 15, a lumen is preferably etched into a substrate 110, such assilicon, using etching techniques such as deep reactive ion etching(DRIE), plasma etching, or any other suitable etching method. This stepis preferably utilized with a mask that covers the portions of thesubstrate 110 to be protected. The walls and associated profiles arethen preferably manufactured through isotropic etching of the substrate110 utilizing a corrosive liquid or plasma, but any other suitableisotropic material removal method can be used. A mask is preferably usedto protect the puncture end. Multiple hollow needles are preferablysimultaneously manufactured as an array 200, but can alternatively beindividually manufactured.

In a second variation of the cell removal tool, the cell removal tool600 is also configured to puncture the top layer 120 from a directionnormal to the broad face of the substrate 112. The cell removal tool 600preferably removes the cell in a substantially normal direction from thebroad face of the substrate 112, but can alternatively remove the cellin an angled direction relative to the broad face of the substrate 112.As shown in FIG. 16, the cell removal tool 600 preferably includes ahollow needle pair including a first needle 640 and a second needle 660,wherein both needles are preferably substantially similar to thatdescribed in the first variation of the cell removal tool 600. The firstand second walls of the first needle 640 are preferably configured toform a first and second fluid impermeable seal with the inlet channel240 and/or the pore chamber 222. The first and second walls of thesecond needle 660 are preferably configured to form a first and secondfluid impermeable seal with the outlet channel 260 and/or pore channel224. The first and second needles are preferably aligned in parallel,with the perforating tips of the first and second needles adjacent andoriented in the same direction within the cell removal tool 600 (e.g.wherein both tips are located on the same side of the cell removal tool600). The first and second needles are preferably manufactured using theaforementioned manufacturing process, but can alternatively bemanufactured using different processes. The first and second needles arepreferably simultaneously manufactured on the same substrate 110, butcan alternatively be separately manufactured and joinedpost-manufacture. The distance between the first and second needle 660is preferably substantially equivalent to the pore 220 length (e.g. thesum of the chamber 222 and pore channel 224 lengths). However, thedistance between the first and second needle 660 can be the chamberlength, the pore channel 224 length, or any suitable distance. The firstand second needles 660 preferably cooperatively form a fluidly isolatedvolume, the fluidly isolated volume including one or more pores ofinterest, segment of the inlet channel 240 adjacent the pore(s) ofinterest, and segment of the outlet channel 260 adjacent the pore(s) ofinterest, such that the pore(s) of interest are fluidly isolated fromadjacent pores 220. In operation, fluid is preferably ingressed throughthe second needle 660 into the fluidly isolated segment of the outletchannel 260, through the pore channel 224, through the chamber 222, andinto the first needle 640. As the fluid moves through the chamber 222,the fluid preferably entrains the retained cell and moves the cell intothe first needle 640. The second needle 660 can be fluidly coupled to apump and a fluid source. Alternatively/additionally, the first needle640 can be fluidly coupled to a low-pressure generator (e.g. a pump).The fluid is preferably a buffer, but can alternatively be cell culturemedia or any other suitable fluid that retains cell viability.

In a third variation of the cell removal tool, the cell removal tool 600is configured to remove one or more cells from the cell capture system100 in a direction substantially parallel to the broad face of thesubstrate 112. As shown in FIG. 17, the cell removal tool 600 preferablyincludes a cannula 680 defining a lumen and an aperture 684. The cannula680 preferably terminates in a sealed puncture tip 682 at a first end,and is preferably fluidly connected to a cell collection volume at asecond end. The aperture 684 is preferably a hole that extends throughthe cannula 680 wall, wherein the hole preferably has a widthsubstantially equivalent to or larger than the width of a pore chamber222, but small enough such that the aperture 684 does not span two porechambers 222. The cannula 680 preferably includes one aperture 684, butcan alternatively include multiple apertures 684, wherein the multipleapertures 684 can be aligned in a line parallel to the longitudinal axisof the cannula 680, or can be distributed about the surface of thecannula 680 (e.g. spiral about the longitudinal axis of the cannula680). The aperture 684 preferably extends through a longitudinal cannula680 wall, but can alternatively extend through a portion of the puncturetip 682. In one example, the aperture 684 extends through a portion ofthe longitudinal cannula wall proximal the puncture tip 682. In anotherexample, the aperture 684 extends through a portion of the longitudinalcannula wall a predetermined distance from the puncture tip 682, whereinthe distance can be configured such that the cannula wall blocks one ormore of the adjacent pores 220. In another example, the aperture 684 canextend through the puncture tip 682 such that the longitudinal axis ofthe aperture 684 extends in parallel or coaxially with the longitudinalaxis of the cannula 680. The transition between the aperture 684 and thecannula 680 exterior and/or interior is preferably convex and curved toprevent cell damage, but can alternatively be concave, angled, be atright angles, or have any suitable configuration. The cannula 680preferably has a circular cross section, but can alternatively have arectangular or square cross section, ovular cross section, or any othersuitable cross section. The cannula 680 is preferably rigid, but canalternatively be flexible or include flexible portions. In onealternative, the cannula 680 is flexible and includes a rigid puncturedevice 686, wherein the rigid puncture device 686 is slidably coupledover the cannula 680. The rigid puncture device 686 forms and retains anentryway into the inlet channel 240, and the cannula 680 can be advancedtherethrough. However, the cannula 680 can have any other suitableconfiguration. The cannula 680 can additionally include a perforatorslidably coupled within the lumen, wherein the perforator can extendthrough the aperture 684 to perforate any intermediary layers betweenthe cannula 680 and the pore 220 (e.g. an isolation layer). Theperforator position post perforation can be retained to facilitate cellremoval therethrough, or the perforator can be retracted prior to cellremoval.

In one variation of cell retrieval tool operation, the cannulapreferably traverses through the inlet channel 240 of an array 200having a cell of interest 10 until the aperture is aligned with the pore220 containing the cell of interest 10. Fluid can then be ingressedthrough the associated outlet manifold 400, wherein the pressure of theingressed fluid pushes the cell of interest 10 out of the pore chamber222, through the aperture, and into the cannula. Subsequent fluidingress through the inlet channel 240 can recapture any cells that werebackflowed out of their respective pores 220. The cannula canadditionally or alternatively include a low-pressure generationmechanism fluidly coupled to the lumen that aspirates the cell out ofthe pore 220. Alternatively or additionally, the cannula can facilitatecell ingress through capillary action. The cell preferably travelsthrough the lumen and is stored within the cell collection volume.

In this variation of cell retrieval tool operation, the cannula ispreferably inserted into the inlet channel 240 through the side of thesubstrate 110, as shown in FIG. 17B, wherein the inlet channel 240preferably partially defined by a self-sealing wall. The cannula ispreferably extended through this self-sealing wall. Alternatively, thecannula can be inserted into the inlet channel 240 through the top layer120, wherein the cannula can be flexible to accommodate the angle ofentry, or the top layer 120 can be elastic to accommodate the angle ofentry. However, any other suitable method of introducing the cannulainto the inlet channel 240 can be used.

In another variation of cell retrieval tool operation, the cannulaincludes an aperture through the puncture tip. The cannula is advancedthrough the inlet channel 240, successively blocking each successivepore chamber 222 until only the desired subset of pores 220 are leftuncovered. Fluid can then be provided through the outlet channel 260directly fluidly connected with the uncovered pores 220 tosimultaneously release the cells from the uncovered pores 220, whereinthe fluid preferably entrains the cells and moves the cells into thecannula. The cannula can additionally or alternatively be fluidlyconnected to a low-pressure generator to aspirate the cells into thecell collection volume.

Cell removal from the cell capture system 100 is preferably automated,but can alternatively be semi-automated or manual. In one variation,cell removal is automated, wherein an integrated platform 30 identifiesand removes the cells of interest. Cell identification can includeautomatic fixing, permeabilzation, staining, imaging, and identificationof the cells through image analysis (e.g. through visual processing witha processor, by using a light detector, etc.). Cell removal can includeadvancement of a cell removal tool 600 to the pore 220 containing thecell of interest 10. Cell removal can additionally include cell removalmethod selection and/or cell removal tool selection. In anothervariation, cell identification can semi-automated, and cell retrievalcan be automated. For example, cell staining and imaging can be doneautomatically, wherein identification and selection of the cells ofinterest can be done manually. In another variation, all steps can beperformed manually. However, any combination of automated or manualsteps can be used.

Example Applications

The cell capture system 100 described above can be used for a variety ofbiological assays and procedures. Running an assay or procedurepreferably includes capturing target cells in addressable locationswithin the cell capture system and delivering reagents to the interioror surface of each captured cell while maintaining cell registrationwith its respective pore or location.

In a first example, the cell capture system 100 can be used as amicroarray 200, wherein microspheres 140 are introduced into the cellcapture system 100 prior to sample introduction. The microspheres 140are preferably slightly larger than the pore channels 224, but canalternatively be smaller. The microspheres 140 can be coated withspecific analytes (e.g. affinity molecules, etc.), wherein themicrospheres 140 can create affinity columns within the pores 220. Themicrospheres 140 can additionally be tagged for imaging. In onevariation, multiple sets of microspheres 140 are sequentially introducedinto the cell capture system 100, wherein each set of microspheres 140has an affinity molecule coating different from the other sets. Eachmicrosphere set is preferably tagged with the same imaging tag (e.g. alltagged with Cal Red), but can alternatively be tagged with differentimaging tags. Each microsphere set preferably includes a small number ofmicrospheres 140 (e.g. less than the number of pores 220 in the system),but can alternatively have more. The cell capture system 100 ispreferably imaged after each microsphere set is introduced to identifythe pores 220 occupied by the constituent microspheres 140 of the set.However, the cell capture system 100 can be imaged after all themicrospheres 140 are introduced, particularly when each microsphere setis tagged with a different image tag. In this way, a highly multiplexedbead microarrays 200 can be created within the cell capture system 100.In another variation, as shown in FIG. 18, the microspheres 140 can formsmall pore networks within the pores 220 that functions as a smallerpore filter devices. For example, microspheres 140 of approximately 10microns can be used to create a bacteria filter, while microspheres 140of approximately 2 microns can be used to create a virus filter. Inanother example, affinity molecule-coated microspheres can be introducedcontemporaneously with the sample, wherein the microspheres bind withthe target cells to form complexes. The microspheres are preferablysized such that the complexes are trapped within the pores while unboundmicrospheres flow through the system. The microspheres 140 can bepolymeric, metallic, paramagnetic, magnetic, or have biologicalproperties. For example, the microspheres 140 can be made of thermallyconductive materials and can function as rapid heat exchanger units.

In another example, one or more assays can be run within the cellcapture system 100. The cells of interest are preferably first isolatedby running the sample through the cell capture system 100. The capturedcells are preferably then stained, wherein staining preferably maintainsthe cell viability. Cell analysis, including morphology and cellcounting, is then preferably performed. One or more assays can then beperformed on the captured cells. These assays may includeImmunocytochemistry, Fluorescence In-situ Hybridization (FISH),Polymerase Chain Reaction (PCR), Enzyme Linked Immunosorbent Assay(ELISA) and other standard cellular and molecular assays known to aperson skilled in the art.

Isolating the cells of interest preferably includes pumping the samplethrough the cell capture system inlet 320 and egressing the remainder ofthe sample through the cell capture system 100 outlet 420. Isolating thecells of interest can additionally include sample enrichment prior tosample ingress into the cell capture system 100. Isolating the cells ofinterest can additionally include running a buffer through the cellcapture system 100 to rinse the isolated cells. Isolating the cells ofinterest preferably includes leaving the cells within the pores 220, butcan alternatively include cell removal from the cell capture system 100.The removed cells can be passed through a second cell capture system 100to sequentially enrich the isolated cell population, or can be storedwithin a cell collection volume for off-chip analysis.

Antibody staining is preferably used to identify the pores 220 thatcontain the cells of interest. Antibody staining can additionallydistinguish the cells of interest over undesired cells 20 of similarsize that have also been captured. Antibody staining preferably includesintroducing a solution of conjugated antibodies, specific to the cell ofinterest 10, through the cell capture system 100. The conjugatedantibodies are preferably primary antibodies, but can alternatively besecondary antibodies, wherein unconjugated primary antibodies arepreferably introduced into the cell capture system 100 prior toconjugated antibody introduction. However, any suitable cell stainingmethod can be used.

Cell analysis is preferably used to determine the morphology of thecaptured cells and to determine the number and location of capturedcells of interest. Cell analysis is preferably performed by anassociated integrated platform 30, wherein morphology and cell countingis preferably accomplished through global chip imaging and imageanalysis. Imaging and analysis is preferably automatically performed,but can alternatively be semi-automated or manually performed. However,morphology determination and cell counting can be achieved through anyother suitable method.

Running assays on the isolated cells functions to determinecharacteristics of the cells and/or determine cell responses to givenstimuli. Analyses can be run on the cells individually (e.g. single celllevel analysis), wherein cells can be individually fluidly isolatedwithin the cell capture system 100. Alternatively, analyses can be runon the cell capture system 100 as a whole. Alternatively, individualarray 200 subsets can be fluidly isolated from other array 200 subsets,wherein different analyses can be performed on different array 200subsets. Example assays that can be run on the cells include FISHassays, selective cell lysing and lysate collection, single cellmolecular analysis (e.g. PCR, RT-PCR, Whole Genome Amplification,ELISPOT, ELISA, Immuno-PCR, etc.), drug testing, cell culturing,affinity analyses, time-responsive analyses, but other analyses canalternatively/additionally be run. Isolated cells can be removed priorto, during, or after the assays have been run, preferably with the cellremoval tool 600 but alternatively with any suitable method.Alternatively, isolated cells can be isolated within the chamber 222(e.g. with an isolation layer), fixed, cultured within the chamber 222,or be retained within the chamber 222 in any other suitable manner.

In one specific example, assaying cells with the cell capture system 100includes pre-processing a sample containing spiked cancer cells, primingthe cell capture system 100, flowing the sample through the cell capturesystem 100, fixing the cells within their respective pores, and stainingthe fixed cells. After the assaying procedure, the cells can be manuallyor automatically imaged and analyzed. The sample is preferably aperipherial whole blood sample, but can be any other suitable samplecontaining target cells. The cell capture system 100 preferably includes12,800 pores, but can alternatively include more or less pores.Pre-processing the sample preferably includes diluting the sample (e.g.with a 0.5% formalin in 1×PBS mixture or any other suitable solutioncontaining a fixing agent) and incubating the sample, preferably in arocker (e.g. for 15-30 minutes). Priming the cell capture system 100preferably includes introducing an initial buffer (e.g. 1% BSA+ 0.1%triton X in 1×PBS) and removing air bubbles from the system 100. Flowingthe sample through the cell capture system 100 preferably includesflowing the sample through the system 100 at a pressure of less than10,000 Pa in less than 10 minutes while minimizing the introduction ofair bubbles, but can alternatively include flowing the sample throughthe system 100 at any suitable pressure in any suitable time frame.Fixing the cells preferably includes post fixing the cells with a fixingagent (e.g. 2% formalin in wash buffer), which can prepare the cells forsubsequent antibody staining. Staining the fixed cells can includewashing the fixed cells (e.g. with 1% BSA+25 mM EDTA in 1×PBS) andintroducing an antibody cocktail containing antibodies specific to thecells of interest (e.g. a primary antibody cocktail includinganti-cytokeratin 8/18 or anti-EpCAM that recognize human epithelialcancer cells, CD45 that recognizes leukocytes, and/or nuclear stainHoescht 33342) into the cell capture system 100. Staining the fixedcells can additionally include incubating the cells (e.g. for 30-45minutes at room temperature). Staining the cells can additionallyinclude washing the cells with a wash buffer (e.g. 1% BSA+25 mM EDTA inIX PBS), introducing a secondary antibody cocktail containing antibodiesthat bind to the primary antibodies (e.g. a cocktail includingAlexa-conjugated anti-CD45, anti-cytokeratin 8/18, and/or anti-EpCAM),and incubating the cells (e.g. at room temperature for 45 minutes).Assaying the cells can additionally include wash steps between eachassay step. The cells are preferably washed with a wash buffer includingculture media, buffer, metal ion scavengers and/or surfactants (e.g. awash buffer including 1% BSA and 0.1% triton X in 1×PBS, a wash bufferincluding EDTA, etc.).

Sample Preparation

The cell capture system 100 is preferably used with a cell-containingsample. The cell-containing sample is preferably a blood sample, but canalternatively be bodily fluid, cells suspended in buffer or culturemedium, or any other suitable cell-containing sample.

While the cell-containing sample can be introduced into the cell capturesystem 100 without any pre-processing, pre-processing can be preferredto increase the efficacy of cell sorting. Sample pre-processingpreferably includes sample enrichment to increase the proportion ofdesired cells 10 within the sample. Sample enrichment preferablyincludes substantially removing undesired components from the samplebefore sample ingress into the cell capture system 100. Samplepre-processing can additionally include preparing the sample fordownstream processing or processing the sample in any other suitablemanner for any suitable application.

In a first variation, sample components that can form obstacles, such asclots, within the cell capture system 100 are preferably removed. Forexample, in a blood sample, such components can include red blood cells,platelets, and other similar blood components. These components arepreferably removed through density gradient centrifugation, wherein theerythrocyte and granulocyte pellet is preferably discarded, and theremainder of the sample retained. However, these components can beremoved through filtration, selective lysing, or any other suitablemethod of removal or inactivation.

In a second variation, undesired cells 20 of substantially the same sizeas the desired cells 10 are selectively removed. For example, if CTCsare the desired cells 10, then mono-nuclear cells (e.g. PMBCs) arepreferably removed. Undesired, similarly-sized cells are preferablyremoved by negative selection, but can alternatively be removed by othersuitable removal methods, such as centrifugation. Negative selection ispreferably achieved through immunomagnetic separation of undesiredcells, wherein antibody-coated magnetic particles are introduced intothe sample. The antibodies coating the magnetic particles are preferablytargeted toward antigens expressed by the undesired cells 20 but notexpressed by the desired cells 10. For example, if leukocytes are theundesired cells 20, then anti-CD45 can be used. The sample is thenpassed through a magnetic field, wherein the magnetic particlesselectively remove the bound, undesired cells 20 from the sample.

Negative selection can alternatively or additionally be achieved withinthe cell capture system 100, wherein the cell capture system 100includes a first stage fluidly connected to a downstream a second stage.The channels of the first stage preferably include affinity molecules(e.g. antibodies) that selectively bind the undesired cells 20, whilepermitting the desired cells 10 to flow therethrough. The affinitymolecules can be introduced as a coating, as affinity molecule-coatedmicrospheres 140, affinity molecule-coated micropillars, affinitymolecule-coated microchannels, or introduced in any other suitablemanner. The first stage can be a portion of the inlet manifold 300 or asubset of upstream arrays 200, a separate cell capture system 100, orany suitable upstream stage. The first stage preferably includes largepore channel size s, preferably larger than the diameter of the desiredcell 10 (e.g. 35-50 micrometers). The second stage preferably selectsfor the desired cell 10 according to cell size and/or deformability, andpreferably does not include any antibodies or cell-binding coatings.

In one variation of sample preparation, as shown in FIG. 19, the sampleis prepared by removing small sample components through density gradientseparation S100 and removing mononuclear cells through immunogenicseparation S200. The cells of interest are then isolated using the cellcapture system S300, and subsequent assays are performed on the isolatedcells S400.

Integrated Platform

As shown in FIG. 20, the cell capture system 100 is preferably utilizedwith an integrated platform 30 including a sample workstation 40 and animaging platform 50. The integrated platform 30 is preferably fullyautomated, but can alternatively be semi-automatic or manually operated.The integrated platform 30 can perform all or some the functions ofpipetting, aliquoting, mixing, pumping, and monitoring. The integratedplatform 30 can additionally automatically identify occupied chambers222, image said chambers 222, and/or perform analyses on said chambers222. The integrated platform 30 can additionally selectively removecells from the cell capture system 100. The integrated platform 30 canadditionally or alternatively perform any other suitable function. Thecell capture system 100 is preferably utilized with a cell capturesystem 100 as described above, but can alternatively be utilized withany suitable apparatus or method.

The sample workstation 40 preferably includes a pumping system thatregulates the sample flow rate through the system to control the shearforces on the cells while providing enough positive pressure to pushunwanted cells and fragments through the pore chambers 222 of the pores220. In one variation, the pumping system provides a pumping pressureless than 10,000 Pa. More preferably, the pumping system provides apumping pressure of approximately 6,000 Pa, but can alternativelyprovide any suitable pumping pressure. The pumping system is preferablycapable of handling varying volume inputs, preferably ranging from 100microliters to tens of milliliters. As shown in FIG. 21, the pumpingsystem preferably couples to the inlet 320 and outlet 420 of the cellcapture system 100 through a fluidic manifold 42, wherein the fluidicmanifold 42 preferably introduces fluid into the cell capture system 100from above, but can alternatively introduce fluid into the cell capturesystem 100 from below, from the side, or from any suitable direction.The fluidic manifold 42 preferably includes fluid seal-forming elements43 about the inlet- and outlet-contacting portions, such as O-rings,gaskets, or any other suitable sealing element. The sample workstation40 preferably includes a venting system to vent air bubbles (e.g. usinghydrophobic vents). The sample workstation 40 can additionally functionto prepare the sample for use with the cell capture system 100. Forexample, the sample workstation 40 can mix reagents, facilitate unwantedcell removal from the sample, or perform any other suitable function.The sample workstation 40 can additionally function to retrieve capturedcells, and can include the cell collection volume and the low-pressuregenerator, if used.

The workstation preferably enables simultaneous processing of multiplesamples (e.g. 12, 24, 96 samples, etc.) of blood or any other suitablespecimen. As shown in FIG. 22, the sample workstation 40 canadditionally include predetermined sample locations, wherein sampletubes, such as specimen tubes, can be loaded into a specific locationfor positive identification throughout the process. Specimen tubes caninclude unique identifiers (e.g. barcodes) that can be automaticallyidentified by the workstation or manually read. The sample workstation40 can additionally accept reagents used to process the sample and/orcaptured cells. The reagents are preferably provided as a unitizedreagent strip containing pre-loaded or partially loaded reagents, butcan alternatively be provided as separate vials or in any other suitableform factor. Reagents can include wash buffers, purge liquids, cellstaining reagents, cell fixing reagents, cell growth media, cell lysingreagents, reagents required for in-situ hybridization, reagents requiredfor specific nucleic acid amplification (e.g. PCR reagents), reagentsrequired for blocking the function of specific moieties, reagentsrequired for cleaning the cell capture system 100, or any other suitablereagent. The configuration of reagents on the strip and/or order ofreagent provision or arrangement is preferably dependent on theprocesses desired. The workstation preferably accepts reagents formultiple processes, wherein multiple processes can be simultaneouslyperformed on a single chip. Examples of processes that can be performedinclude immunostaining, single cell proteomic analysis, nucleic acidanalysis, genomic sequencing, or a comparison between the expressed cellRNA and the background plasma expression, testing the efficacy ofpharmaceutical agents. The sample workstation 40 is preferablycontrolled by an independent processor, but can alternatively becontrolled by any suitable control mechanism.

The integrated platform 30 can additionally include an imaging platform50. The imaging platform 50 can function to capture images of cells. Thedigital imaging system can additionally include software that can allowfor specific image quantization and reporting in the platform. Theimaging platform 50 preferably includes imaging hardware and imagingsoftware. The imaging software preferably controls the imaging hardware,and can additionally process the images. In one variation, the imagingsoftware analyzes a first image to determine addresses of the pores 220retaining cells of interest, then controls the imaging hardware toindividually image and interrogate each identified pore 220. The imagingsoftware can additionally store the location of the cells of interestfor further cell processing, such as cell removal or single cellanalysis.

The imaging hardware is preferably configured to accept the cell capturesystem 100, and can additionally accept conventional imaging equipment,such as microscope slides, cell culture plates, or any other suitableimaging equipment. The imaging hardware is preferably capable ofauto-focusing the microscope before image capture, but can alternativelytake a series of images at multiple focal lengths, use imagepost-processing to sharpen the image, or utilize any other suitablemethod to achieve a focused image.

The imaging hardware preferably includes an automated stage 52 that canfacilitate self-calibration, cell capture system interrogation, cellcapture system 100 agitation, or move the imaging equipment in any othersuitable manner. The automated stage 52 can additionally function toalign the cell capture system 100 with the objective or field of image.The automated stage 52 can additionally move the cell capture system 100relative to the cell removal tool 600 to align the cell removal tool 600aperture with a desired pore 220. The automated stage 52 canadditionally move the cell to the sample workstation 40. The automatedstage 52 is preferably driven by a motor, but can be driven by any othersuitable mechanism.

The automated stage 52 is preferably capable of moving in at least thez-direction, and can additionally move in the x-direction and/ory-direction. In one variation, as shown in FIG. 23, the stage isadditionally capable of tilting the cell capture system 100, which canenable imaging module 56 autofocus. The cell capture system 100 can betilted at a specified angle, wherein some areas of the slide image willbe in better focus than others based on the different resultant focallengths. The contrast differences created are then interrogated by acomputer algorithm that determines the vertical section with thegreatest contrast, a measure indicative of the ideal focal length(optimal z-height). The automated stage 52 then replaces the cellcapture system 100 to a flat position parallel the base, and move thecell capture system 100 to the determined optimal z-height.

The stage preferably includes a retention mechanism 54 that retains thecell capture system 100 position relative to the rest of the stage. Theretention mechanism 54 is preferably further capable of retaining otherimaging equipment, such as glass slides or cell culture plates. Theretention mechanism 54 can be as a clip that biases the cell capturesystem 100 against a brace, a recess in a broad face of the stage, orany other suitable retention mechanism 54. The stage preferablyaccommodates one cell capture system 100 at a time, but canalternatively accommodate multiple cell capture system 100 ssimultaneously. In one variation, the stage includes a carousel orconveyor tray that includes a plurality of cell capture system 100 s,wherein the stage rotates successive cell capture system 100 s under theimaging module 56.

The stage can additionally include a thermal control system thermallycoupled to the portion of the stage configured to contact the cellcapture system 100. The thermal control system can be used to controlthe cell capture system 100 temperature by heating and/or cooling thecell capture system 100 during assays or reactions. For example, thethermal control system can heat the cell capture system 100 to incubatethe cells retained therein, and cool the cell capture system 100 toquench given biochemical reactions. In one variation, the thermalcontrol system includes a single block configured to contact an entirebroad face of the cell capture system 100. In another variation, thethermal control system includes multiple sections, each sectionconfigured to heat or cool a given portion of the cell capture system100 broad face. The thermal control system preferably includes electricheaters, but can alternatively include inductive heaters, ceramicheaters, or any other suitable heaters. The thermal control system caninclude a heat sink, heat pump, heat exchanger, or any other suitablepassive or active cooling mechanism. The thermal control system ispreferably optically transparent, but can alternatively have any othersuitable optical property.

The stage can additionally include a fluidic manifold 42 that interfaceswith the inlet 320 and outlet 420 of the cell capture system 100, suchthat real-time flow through the cell capture system 100 can bevisualized.

The imaging hardware preferably additionally includes an imaging module56 including an imager and an optimized illuminator capable of capturinghigh-resolution images at multiple predefined locations of the slideand/or global images of the slide. The imaging module 56 is preferablycapable of working with various sets of emission and excitationwavelengths, such that the imaging platform 50 can resolve multiplemarkers (e.g. fluorescent markers, stains, etc.). The illuminator ispreferably capable of providing the appropriate illumination andwavelengths for fluorescence resolution, phase contrast microscopy,dark-field microscopy, bright-field microscopy, and/or any othersuitable imaging technique. For example, the imaging hardware caninclude one or more emitters capable of resolving fluorescence dyes suchas FAM, Cal Red, Texas Red, Cy5, CY5.5, or any other suitablefluorescence dye used in cell analysis. The imaging module 56 preferablyincludes an imager that is preferably optically connected to amicroscope that magnifies the portion of the cell capture system 100 tobe imaged. The imager can be 5 megapixel, 10 megapixel, 20 megapixel, 50megapixel, 100 megapixel, or any suitable size imager. The imager can bea CCD, CMOS imager, line scanner, or any other suitable imager.

The imaging hardware can additionally include an identifier reader thatfunctions to read and identify imaging equipment identifiers. Theimaging hardware can include a barcode reader, a RFID tag reader, a QRcode reader, a nearfield communication device, or any other sutiablemechanism that can identify a unique identifier located on the imagingequipment (e.g. the cell capture system 100, a microscope slide, etc.).Alternatively or additionally, the imaging module 56 can be used as theidentifier reader. In one variation, a given objective lens is placedover the unique identifier to obtain the correct aspect ratio forimaging module 56 imaging. In another variation, the unique identifiercan be pieced together from multiple images. However, any other suitablemethod of obtaining and identifying the unique identifier can be used.

The imaging hardware is preferably controlled by a processor runningimaging software, wherein the processor preferably controls stagemotion, microscope focus, and image capture, and can additionallycontrol other functions. Imaging hardware control is preferably based onan image taken by the image hardware, but can alternatively be based onsignals received from sensors or other portions of the integratedplatform 30.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

What is claimed is:
 1. A system comprising: a substrate defining a setof chambers, the set of chambers configured to retain individual targetsof a set of targets of a sample and each of the set of chamberscomprising: one or more walls defining a chamber volume, and an opensurface permitting access of an individual target of the set of targetsto the chamber volume from a direction perpendicular to the broadsurface of the substrate, wherein the substrate is thermally conductive,and wherein contents of each chamber of the set of chambers areconfigured to receive heat through the substrate; an inlet channelpositioned upstream of the set of chambers and fluidly coupled to theset of chambers; and an outlet channel positioned downstream of the setof chambers and fluidly coupled to the set of chambers, wherein fluidfrom the inlet channel reaches the outlet channel only by way of the setof chambers.
 2. The system of claim 1, wherein the substrate provides athermal interface to a thermal control system comprising a heatingelement.
 3. The system of claim 1, wherein the set of chambers of thesubstrate is configured to provide environments for performing apolymerase chain reaction (PCR)-associated assay.
 4. The system of claim1, further comprising a vent.
 5. The system of claim 1, furthercomprising a seal configured seal at least a portion of the substrate.6. The system of claim 1, wherein the set of chambers comprises a set ofchamber arrays arranged in parallel, each of the set of chamber arrayscoupled at an upstream end to the inlet channel and coupled at adownstream end to the outlet channel.
 7. The system of claim 1, furthercomprising an inlet configured to receive the sample and direct thesample into the inlet channel.
 8. The system of claim 1, wherein the setof targets comprises cell-derived material, and wherein the set ofchambers is configured to provide environments for performing at leastone of a cell processing operation and a molecular reaction.
 9. Thesystem of claim 1, wherein a characteristic dimension of each chamber isless than 200 micrometers.
 10. The system of claim 1, wherein the set ofchambers comprises from 1,000 to 1,000,000 individual chambers.
 11. Amethod comprising: providing a substrate comprising an inlet channel, anoutlet channel, and a set of chambers in fluid communication with theinlet channel and the outlet channel, wherein flow from the inletchannel is configured to reach the outlet channel only upon passing theset of chambers; receiving a fluid sample comprising a set of targetsinto the inlet channel; capturing and partitioning the set of targets,by way of the set of chambers; and providing an environment forconducting a set of processes at the substrate, wherein the set ofprocesses comprises an amplification process configured foramplification of nucleic acid material of the set of targets.
 12. Themethod of claim 11, wherein providing the environment for theamplification process comprises receiving polymerase chain reaction(PCR) reagents into the inlet channel.
 13. The method of claim 11,wherein the substrate provides a thermal interface to a thermal controlsystem comprising a heating element, and wherein performing theamplification process comprises heating contents of the set of chambersthrough the substrate.
 14. The method of claim 11, wherein the set ofprocesses comprises sample processing for at least one of a proteomicanalysis and a genomic analysis.
 15. The method of claim 11, furthercomprising forming a seal between the substrate and a seal-formingmaterial, thereby sealing contents of the substrate.
 16. The method ofclaim 11, further comprising venting contents of the substrate duringperformance of one or more of the set of processes.
 17. The method ofclaim 12, wherein receiving the fluid sample comprises pumping the fluidsample into an inlet and directing the fluid sample from the inlet andinto the inlet channel of the substrate.
 18. The method of claim 11,wherein the set of processes comprises a multiplexed sample processingoperation across the set of targets of the fluid sample.
 19. The methodof claim 11, further comprising scanning a barcode of the substrate incoordination with receiving the fluid sample at the substrate andperformance of the set of processes.
 20. The method of claim 11, whereinthe set of chambers comprises a set of chamber arrays arranged inparallel, each of the set of chamber arrays coupled at an upstream endto the inlet channel and coupled at a downstream end to the outletchannel, and wherein receiving the fluid sample comprises delivering thefluid sample into the set of chamber arrays in parallel.